User:Karmella Haynes/Notebook/BioBrick cloning/2011/06/01

Project name

Main project page Previous entry      Next entry

06/01/11 ✓ Cultures/minipreps/digests: assemblies from 5/31/11 (2 each) ✓ Assemblies: re-do KAH188, 190; new KAH191, 195, 196, 197, 198 Minipreps > Check with E/P digests Reagent Volume Expected: 1,2. KAH185 = 542 3,4. KAH186 = 2025 5,6. KAH187 = 2269 7,8. KAH188 = 2124 9,10. KAH189 = 2296 11,12. KAH190 = 2368 13,14. KAH192 = 2461 15,16. KAH193 = 2788 17,18. KAH194 = 3032 15 μL/lane; 1% agarose DNA(plasmid) 2.0 μL 10X buffer 1.5 EcoRI 1.0 PstI 1.0 dH2O 9.5   15 μL --> 37°C/ ~15 min. --> Re-do KAH188, 190 (to get better cloning ratio, less background, use less vector) Assemblies KAH188: KAH83/(E/S)/443 ✓ + KAH182/(E/X)/~4875 ✓ KAH190: KAH85/(E/S)/687 ✓ + " KAH191: (1) KAH185/(E/S)/size + " KAH195: (2) KAH193/(S/P)/~5988 + polyA2/(X/P)/225 ✓ KAH196: " + KAH171/(X/P)/418 ✓ KAH197: (3) KAH194/(S/P)/~6232 + polyA2/(X/P)/225 ✓ KAH198: " + KAH171/(X/P)/418 ✓ > Digests (Fermentas FD) --> Specific notes Reagent Volume   30 μL/lane, 1% agarose DNA (plasmid) up to 25 μL 10x buffer 3.0 enzyme 1 1.0 enzyme 2 1.0 dH2O ---   30 μL --> 37°C/ ~30 min. > Measure conc.'s Sample OD260 260/280 ng/μL 1. KAH185 (E/S) 0.198 2.03 9.9 2. KAH193 (S/P) 0.188 1.74 9.4 3. KAH194 (S/P) 0.178 1.99 8.9 > Ligations Ligation Plate results (lig : neg crtl) mm/dd/yy 1. KAH83(E/S)/443, 1 ng + KAH182(E/X)/4875, 5 ng KAH188 2:1 (Pick 2) 2. KAH85(E/S)/687, 2 ng + " KAH190 2:1 (Pick 2) 3. KAH85(E/S)/687, 2 ng + " KAH191 1:1 (Pick 4) 4. KAH182(E/X)/ 5 ng lots of colonies, even with only 0.5 DNA 5. polyA2(X/P)/225, 1 ng + KAH193(S/P)/5988, 10 ng KAH195 >10:1 (Pick 2) 6. KAH171/(X/P)/418, 1 ng + " KAH196 >10:1 (Pick 2) 7. KAH193(S/P)/ 5 ng   5. polyA2(X/P)/225, 1 ng + KAH194(S/P)/6232, 10 ng KAH197 >10:1 (Pick 2) 6. KAH171/(X/P)/418, 1 ng + " KAH198 >10:1 (Pick 2) 7. KAH193(S/P)/ 5 ng     1 2 3 4 5 6 7 8 9 10 Insert DNA 3.5 3.5 3.5 --- 2.0 2.0 --- 2.0 2.0 --- Vector DNA 0.5 0.5 0.5 0.5 2.0 2.0 2.0 2.0 2.0 2.0 2x lgn buf (Roche) 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 T4 ligase (NEB) 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 dH2O --- --- --- 3.5 --- --- 2.0 --- --- 2.0   10 μL 10 μL 10 μL 10 μL 10 μL 10 μL 10 μL 10 μL 10 μL 10 μL --> Add total lign. to 30 μL DH5α-turbo